Assessment of the tocolytic nifedipine in preclinical primary models of preterm birth

Abstract

Spontaneous preterm birth is the leading cause of perinatal morbidity and mortality. Tocolytics are drugs used in cases of imminent preterm birth to inhibit uterine contractions. Nifedipine is a calcium channel blocking agent used to delay threatened spontaneous preterm birth, however, has limited efficacy and lacks preclinical data regarding mechanisms of action. It is unknown if nifedipine affects the pro-inflammatory environment associated with preterm labour pathophysiology and we hypothesise nifedipine only targets myometrial contraction rather than also mitigating inflammation. We assessed anti-inflammatory and anti-contractile effects of nifedipine on human myometrium using in vitro and ex vivo techniques, and a mouse model of preterm birth. We show that nifedipine treatment inhibited contractions in myometrial in vitro contraction assays (P = 0.004 vs. vehicle control) and potently blocked spontaneous and oxytocin-induced contractions in ex vivo myometrial tissue in muscle myography studies (P = 0.01 vs. baseline). Nifedipine treatment did not reduce gene expression or protein secretion of pro-inflammatory cytokines in either cultured myometrial cells or ex vivo tissues. Although nifedipine could delay preterm birth in some mice, this was not consistent in all dams and was overall not statistically significant. Our data suggests nifedipine does not modulate preterm birth via inflammatory pathways in the myometrium, and this may account for its limited clinical efficacy.

Full text

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Assessment of the tocolytic
nifedipine in preclinical primary
models of preterm birth
Bridget M. Arman
1,2, Natalie K. Binder
1,2, Natasha de Alwis
1,2, Sally Beard
1,2,
Danielle A. Debruin
3,4,5, Alan Hayes
3,4,5, Stephen Tong
2, Tu’uhevaha J. Kaitu’u‑Lino
2 &
Natalie J. Hannan
1,2*
Spontaneous preterm birth is the leading cause of perinatal morbidity and mortality. Tocolytics
are drugs used in cases of imminent preterm birth to inhibit uterine contractions. Nifedipine is a
calcium channel blocking agent used to delay threatened spontaneous preterm birth, however, has
limited ecacy and lacks preclinical data regarding mechanisms of action. It is unknown if nifedipine
aects the pro‑inammatory environment associated with preterm labour pathophysiology and we
hypothesise nifedipine only targets myometrial contraction rather than also mitigating inammation.
We assessed anti‑inammatory and anti‑contractile eects of nifedipine on human myometrium
using in vitro and ex vivo techniques, and a mouse model of preterm birth. We show that nifedipine
treatment inhibited contractions in myometrial in vitro contraction assays (P = 0.004 vs. vehicle
control) and potently blocked spontaneous and oxytocin‑induced contractions in ex vivo myometrial
tissue in muscle myography studies (P = 0.01 vs. baseline). Nifedipine treatment did not reduce gene
expression or protein secretion of pro‑inammatory cytokines in either cultured myometrial cells or
ex vivo tissues. Although nifedipine could delay preterm birth in some mice, this was not consistent in
all dams and was overall not statistically signicant. Our data suggests nifedipine does not modulate
preterm birth via inammatory pathways in the myometrium, and this may account for its limited
clinical ecacy.
Globally, prematurity is the leading cause of neonatal morbidity and mortality, with an estimated 15 million
babies born preterm each year1. Prolongation of pregnancies with threatened spontaneous preterm delivery is
vital, particularly in very early gestation, as each completed week in utero corresponds to signicantly improved
fetal outcomes2. Ideally, the aim of inhibiting spontaneous preterm birth is to prolong pregnancies until they are
closer to term. However this is rarely attainable with the current panel of therapeutics available.
Spontaneous preterm labour is primarily treated with drugs, known as tocolytics, that inhibit uterine contrac-
tions. ere are several choices of tocolytics, with each targeting a dierent mechanism of uterine contraction,
however there is no strong evidence that any tocolytic improves neonatal outcomes3. Realistically, the administra-
tion of these drugs only delays delivery with enough time for corticosteroid treatment for fetal lung maturation,
magnesium sulphate for fetal neuroprotection (in some instances), antibiotics in the case of infection, and for
transport of the patient to an appropriate tertiary hospital.
Nifedipine is one of the most widely used tocolytics. Conventionally used as an anti-hypertensive agent, it
was repurposed as a tocolytic in the 1980s due to its calcium channel blocking abilities4. Nifedipine is an L-type
voltage-gated calcium channel antagonist. It inhibits the inux of extracellular calcium into smooth muscle cells,
preventing calcium-dependent contractions5. Despite nifedipine being considered the standard course of treat-
ment for preterm labour, clinical trials evidence supporting the benet of nifedipine is limited6.
Before transition to clinical use in the 1980s, there was only a small number of preclinical studies investigating
nifedipine as a tocolytic that preceded trials in patients4,7,8. ese few studies were limited, assessing nifedipine’s
ability to reduce uterine contractile activity, therefore much remains unknown about the mechanism of action
OPEN
1Therapeutics Discovery and Vascular Function Group, Department of Obstetrics and Gynaecology, University of
Melbourne, Mercy Hospital for Women, 163 Studley Rd, Heidelberg, Victoria 3084, Australia. 2Mercy Perinatal,
Mercy Hospital for Women, Heidelberg 3084, Australia. 3Institute for Health and Sport, Victoria University,
Melbourne, Victoria 3000, Australia. 4Australian Institute for Musculoskeletal Science, Victoria University, St
AlbansVictoria 3021, Australia. 5Department of Medicine—Western Health, Melbourne Medical School, University
of Melbourne, St Albans, Victoria 3021, Australia. *email: nhannan@unimelb.edu.au
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nifedipine works through. Moreover, there is a lack of human invitro data to determine how nifedipine functions
at the cellular level in the mitigation of preterm labour. Importantly, it is unknown if nifedipine has benecial
actions beyond calcium channel inhibition.
Nifedipine, as well asother tocolytics, work by supressing myometrial contractility, the consequence rather
than the cause of preterm labour. It is unknown whether these tocolytics have an impact on the upstream inam-
matory mediators implicated in the pathophysiology of preterm labour. Importantly, the initiation of labour is
complex, involving a milieu of uterine pro-inammatory cytokine and chemokine signalling at both term and
preterm gestations9. Normal healthy labour is characterised by leukocyte inux, release of pro-inammatory
cytokines such as interleukin (IL)-1B, IL-6, CXC motif chemokine ligand (CXCL)-8 and tumor necrosis fac-
tor (TNF), and a decrease of anti-inammatory agents in the uterus9. Spontaneous preterm labour is caused
by similar inammatory processes but at a pathological level of inammation10,11. Pathogens or danger signals
bind to and activate toll-like receptors in the uterus which triggers a downstream pro-inammatory cascade
culminating in activation of myometrial contractility, rupture of membranes, and cervical remodelling, ulti-
mately leading to preterm labour10–12. It is unknown if nifedipine has anti-inammatory eects that can act on
mitigating these upstream events. Such knowledge could be used to increase the ecacy of tocolytic compounds.
While it is believed that the primary mechanism of nifedipine in the uterus is to prevent calcium-dependent
muscle contractions, there is evidence in non-gestational tissues that nifedipine may exert anti-inammatory and
cytoprotective actions beyond inhibition of calcium channels13,14. erefore, we aimed to test whether nifedipine
demonstrated similar actions in the myometrium.
e current study employed an innovative pipeline of human invitro and exvivo functional models of myo-
metrial contraction and inammation, and a mouse model of preterm birth, to assess the actions of nifedipine
on myometrium.
Results
Human myometrial tissue strip contractility. We rst assessed exvivo myometrial tissue contractil-
ity via organ bath myography to determine the eect of nifedipine on non-labouring human myometrial tissue
(collected at caesarean section; n = 3). Nifedipine treatment signicantly inhibited baseline spontaneous con-
traction frequency in myometrial strips (Fig.1A lower panel, B; P = 0.001). Where contractions did occur in the
post-treatment period aer nifedipine administration, the peaks were signicantly reduced in amplitude, time
to peak, duration, and speed compared with vehicle (Fig.1C–F, all P < 0.01). Vehicle control treatment had no
eect on contraction frequency (Fig.1A upper panel), nor on other measures of contractility (amplitude, time to
peak, duration of single contractions, and speed; Supplementary Fig.S1). Importantly, we assessed myometrial
contraction at the end of assessment using high potassium solution which causes maximal inux of calcium
ions into the cells and is used to test tissue integrity. Tissue was responsive to challenge at the completion of the
experiment, indicating the tissue was functionally active (not fatigued) aer the 4–5h in the myograph cham-
bers (SupplementaryFig.S2).
Myometrial strips collected from another cohort of patients (n = 4) were treated with oxytocin in the myo-
graph tissue baths to further induce contractions. Still in the presence of oxytocin, the myometrial strips were
treated with either vehicle control or nifedipine for one hour. Vehicle control did not alter the frequency of
oxytocin-induced contractions (Fig.2A upper panel), but treatment with nifedipine reduced them (Fig.2A lower
panel, B; P = 0.01). Similarly, nifedipine signicantly reduced the other measures of contractility (amplitude,
time to peak and duration) compared with vehicle control (Fig.2B–E, all P < 0.05). ere was no statistically
signicant dierence in speed of contractions between vehicle- and nifedipine-treated tissue as the variation
within the nifedipine-treated samples was high (Fig.2F).
Human myometrial cell contraction assay. We then assessed whether nifedipine inhibits myometrial
contraction in the collagen-myometrial cell contraction assay over a longer period of time. We rst established
that the basal level of contraction inherent to the cells was a 6.3 ± 1.0%decrease in collagen gel disc size aer
48h,compared to the baseline gel size at 0h (SupplementaryFig.S3).Treatment with nifedipine alone did not
alter the rate of these basal contractions (Supplementary Fig.S4). We next examined whether nifedipine treat-
ment altered collagen-cell contractility within a TNF/LPS-stimulated inammatory environment. Treatment
with TNF/LPS increased collagen-cell disc contractility compared to thebasal contraction rate ofcontrol-treated
cells, with a mean 13.6 ± 0.9% decrease in gel size 48h aer treatment (SupplementaryFig.S3; P = 0.01).Co-
treatment with nifedipine demonstrated inhibition of TNF/LPS induced contraction (SupplementaryFig.S3;
P = 0.004), maintaining a level of contraction similar to the basal vehicle control conditions at 48h (Supplemen-
taryFig.S3; P = 0.66).
Human myometrial cell inammatory response. Aer establishing that nifedipine inhibited contrac-
tion, we next assessed whether nifedipine altered inammatory pathways. Human myometrial cells (cell line)
were stimulated with pro-inammatory agents TNF or LPS.
Treatment with TNF caused a clear upregulation in expression of pro-inammatory cytokine genes: IL-1B
(P < 0.001), IL-6 (P < 0.0001), and CXCL8 (P < 0.0001) compared with vehicle control treatment (Fig.3A–C).
However, co-treatment with nifedipine did not attenuate this elevated gene expression (Fig.3A–C).
Similarly, stimulation of myometrial cells with LPS induced a signicant elevation of IL-1B, IL-6, and CXCL8
mRNA transcripts compared with vehicle control (P < 0.0001, Fig.3D–F). Co-treatment with nifedipine did not
inhibit this increased expression (Fig.3D–F). Cell viability was not aected by any of these treatments (Sup-
plementary Fig.S5).
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Figure1. Myograph data showing anti-contractile eects of nifedipine (0.1µM)on strips of human pregnant
non-labouring myometrium exvivo. (A) Representative trace in the upper panel showing that vehicle control
(VEH)has no eect on established spontaneous contractions. In the lower panel, nifedipine (NIF)diminishes
the established pre-treatment contractions. e y-axis is the measured force (millinewtons; mN). e
entireinitial 120min equilibration period is not shown in these representative traces. (B–F) Nifedipine
signicantly reduces contraction frequency (B), amplitude (C), time to peak (D), contraction duration (E), and
contraction speed (F) (n = 3 patients). Data are presented as percentage of baseline (pre-treatment) contractility.
Each point represents the mean of duplicates. Data were assessed for statistical dierences using t tests. e error
bars represent SEM, ** indicates P < 0.01, and *** indicates P<0.001.
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Figure2. Myograph data showing anti-contractile eects of nifedipine(0.1µM)on strips of human pregnant
non-labouring myometrium exvivo aer incubation with oxytocin (1nM). (A) Representative traces
showing that vehicle control (VEH)does not aect frequency of established oxytocin-induced contractions,
but nifedipine (NIF)signicantly reduces the contraction frequency. e y-axis presents the measured force
(millinewtons; mN). e entireinitial 120min equilibration period is not shown in these representative traces.
(B–F) In the presence of oxytocin, nifedipine signicantly reduces contraction frequency (B), amplitude (C),
time to peak (D), and contraction duration (E) but not contraction speed (F) (n = 4 patients). Each point
represents the mean of duplicates. Data were assessed for statistical dierences using t tests. e error bars
represent SEM, * indicates P < 0.05,** indicates P<0.01, *** indicates P<0.001, and ns denotes no statistical
dierence.
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We next assessed the secretion of cytokines into the conditioned media from the treated myometrial cells.
Treatment with either TNF (Fig.4A–D) or LPS (Fig.4E–H) induced a signicant increase in secretion of soluble
MCP-1, MCP-3, IL-6, and CXCL8 from the cells compared with control (all P < 0.05). However, there was no
dierence in secretion of pro-inammatory cytokines with nifedipine treatment (Fig.4). IL-1B concentrations
were below the level of detection of the multiplex assay.
Human myometrial tissue inammatory response. We next investigated if nifedipine treatment
inhibits the inammatory response in cultured whole human myometrial tissue. Treatment with TNF increased
IL-1B (P = 0.003), IL-6 (P = 0.008) and CXCL8 (P = 0.002) gene expression compared with vehicle control-treat-
ment (Fig.5A–C). Addition of nifedipine did not reduce this increased expression of IL-1B, IL-6, or CXCL8
(Fig.5A–C).
Treatment of myometrial tissue with LPS induced an increase of IL-1B (P < 0.0001), IL-6 (P = 0.0004) and
CXCL8 (P = 0.0003) mRNA expression compared with vehicle control treatment (Fig.5D–F). However, addi-
tion of nifedipine did not reduce this upregulation of IL-1B, IL-6, or CXCL8 (Fig.5D–F). Co-treatment with the
combination of TNF and LPS signicantly increased IL-1B, IL-6, and CXCL8 mRNA expression compared with
control (P < 0.0001, Fig. 5G–I). However, consistent with the previous ndings with the myometrial cell line,
addition of nifedipine did not reduce this upregulation (Fig.5G–I).
We also investigated if nifedipine could alter the secretion of pro-inammatory cytokines from myometrial
tissue. MCP-3 secretion was below the detectable limit for all experiments. Treatment of myometrial tissue with
TNF did not increase secretion of MCP-1, IL-1B, IL-6, or CXCL8 compared with vehicle control treated tissue
(Fig.6A–D). ere was also no dierence in secretion of these pro-inammatory cytokines with concomitant
nifedipine treatment compared with control (Fig.6A–D).
Treatment with LPS induced a signicant increase in secretion of IL-1B (P = 0.002), IL-6 (P = 0.04), and
CXCL8 (P = 0.01) (Fig.6E–H) compared with control-treated tissue, but LPS treatment did not increase secretion
of MCP-1 (Fig.6). Addition of nifedipine did not alter secretion of MCP-1, IL-1B, IL-6, and CXCL8 induced by
LPS treatment (Fig.6E–H).
Figure3. Gene expression of pro-inammatory cytokines by human myometrial cells aer treatment with
vehicle control (VEH), TNF, LPS, and nifedipine (NIF). Treatment of myometrial cells with (A–C) TNF (0.1ng/
ml) and (D–F) LPS (100ng/ml) induces an increase in pro-inammatory mRNA expression of IL-1B, IL-6, and
CXCL8.Addition of nifedipine(10µM)does not reduce this upregulation. Data are presented as fold change
calculated relative to that of TNF-treated cells (A–C) or LPS-treated cells (D–F). Treatments were performed in
duplicate and individual data points represent the mean of those technical replicates (n = 4 experiments (A–C)
and n = 5 experiments (D–F)). Data were assessed for statistical dierences using one-way ANOVA followed by
Dunnett’s multiple comparisons performed on the deltaCt (Ct of the gene of interest subtracted from the Ct of
the YHWAZ reference gene).e error bars represent SEM, *** indicates P < 0.001, **** indicates P < 0.0001, and
ns indicates no statistical dierence.
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Figure4. Pro-inammatory cytokines and chemokines secreted by human myometrial cells were assessed
in media collected aer treatment with vehicle control (VEH), TNF, LPS, and nifedipine (NIF). Treatment of
myometrial cells with (A–D) TNF (0.1ng/ml) and (E–H) LPS (100ng/ml) induces a signicant increase in
production and release into culture media of pro-inammatory cytokines and chemokines MCP-1, MCP-3,
IL-6, and CXCL8 and co-treatment with nifedipine(10µM)does not reduce this increased expression. Data is
expressed as the median uorescence intensity of technical replicates as measured by Luminex assay. Statistical
analysis was performed on the log2 transformation of the uorescence intensity using an ANOVA followed by
Tukey’s multiple comparisons. Treatments were performed in duplicate and individual data points represent the
mean of those technical replicates (n = 4 experiments). e error bars represent SEM, ** indicates P < 0.01, ***
indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no statistical dierence.
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Similarly, co-treatment with both TNF and LPS did not induce increased secretion of MCP-1 compared
with control-treated tissue, but caused a signicant increase in IL-1B (P = 0.0003), IL-6 (P = 0.02), and CXCL8
(P = 0.01) secretion compared with control-treated tissue (Fig.6I–L). ere was no change in the secretion of
these cytokines with nifedipine co-treatment (Fig.6I–L).
Mouse preterm birth study. We nally assessed whether nifedipine treatment could prevent preterm
birth in mice (Fig.7A). In pregnant mice, intraperitoneal administration of LPS on D16.5 induced preterm
birth (within 24h) in all mice, compared to the PBS control mice (normal pregnancy) which all delivered at
term (D19.5, Fig.7B). When the mice were treated with nifedipine at either 1mg/kg or 10mg/kg, there was no
overall signicant reduction in preterm birth. However, nifedipine treatment did prolong pregnancy in two mice
(treated with 1mg/kg nifedipine) by 12h and one mouse (treated with 10mg/kg nifedipine) by 24h, but this
eect was not statistically signicant (Fig.7B).
We also assessed whether the uteri post-delivery had altered expression of genes associated with inammation
or uterine contraction. ere were no dierences in expression of Il-1b, Il-6, Tnf, NLR family pyrin domain con-
taining 3 (Nlrp3), gap junction alpha-1 (Gja1), oxytocin receptor (Oxtr), and prostaglandin-endoperoxide synthase
2 (Ptgs2) between vehicle-treated mice, nifedipine-treated mice with no delay in delivery, and nifedipine-treated
Figure5. Gene expression of pro-inammatory cytokines by human myometrial tissue aer treatment with
vehicle control (VEH), TNF, LPS and nifedipine (NIF). Treatment of myometrial tissue pieces with (A–C)
TNF (1ng/ml), (D–F) LPS (5ng/ml) or (G–I) concurrentTNF and LPS treatment increases pro-inammatory
mRNA expression of IL-1B, IL-6, and CXCL8.Addition of nifedipine (10µM)does not reduce this upregulation.
Data are presented as fold change calculated relative to that of TNF-treated tissue (A–C), LPS-treated tissue
(D–F), or combined TNF/LPS-treated tissue (G–I). Treatments were performed in duplicate and individual
data points represent the mean of those technical replicates (n = 4 experiments). One-way ANOVA followed by
Dunnett’s multiple comparisons was performed on the deltaCt (Ct of the gene of interest subtracted from the
Ct of the TOP1 reference gene).e error bars represent SEM, ** indicates P < 0.01, *** indicates P < 0.001, ****
indicates P < 0.0001, and ns indicates no statistical dierence.
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mice with delayed delivery (gestation longer than 17.5days) (Supplementary Fig.S6). However, C–C motif
chemokine ligand 2 (Ccl2) expression was lower in nifedipine-treated mice that had a delayed delivery compared
with vehicle-control mice and with nifedipine-treated mice that had a preterm delivery (Supplementary Fig.S6F).
Discussion
is study used a novel pipeline of invitro, ex vivo, and murine invivo functional models of myometrial contrac-
tion and inammation to assess the actions of nifedipine on myometrium. We have shown that nifedipine at these
selected doses reduces inammation-induced, oxytocin-induced, and spontaneous myometrial contractions in
human preclinical models. However, nifedipine does not reduce inammatory marker expression or secretion of
inammatory cytokines from human myometrial cells and tissues. Additionally, we have shown that nifedipine
is unable to consistently delay delivery in a LPS mouse model of preterm birth.
Here we have demonstrated that nifedipine acutely inhibited myometrial contractions and maintains pro-
longed inhibition in isolated myometrial cells. e eect of nifedipine was potent and near instantaneous. ese
results support initial preclinical studies showing that nifedipine reduced invitro myometrial contractility (spon-
taneous and oxytocin-induced) and invivo uterine activity in non-pregnant and pregnant patients4,7,8. However,
those previous preclinical studies assessed the ability of nifedipine to reduce uterine contractions but did not
examine any other mechanisms of actions or eects of nifedipine on the myometrium, particularly associated
with inammatory pathways. Here, our study provides a more thorough evaluation of nifedipine, particularly
on the key inammatory response that underpins preterm birth pathophysiology.
In our study, we assessed whether myometrial contraction can be induced in response to pro-inamma-
tory agents, TNF and LPS. Indeed, we were able to demonstrate that mimicking a pathological inammatory
Figure6. Treatment of human myometrial tissue with (A–D)TNF (1ng/ml), (E–H) LPS (5ng/ml), and
(I–L) concurrentTNF and LPS, and co-treatment with nifedipine (NIF). TNFdoes not induce secretion
of MCP-1 (A), IL-1B (B), IL-6 (C) or CXCL8 (D) compared with vehicle control (VEH). Addition of
nifedipine(10µM)does not have any eect whencompared with vehicle. LPS does not increase MCP-1
secretion (E), but does induce a signicant increase in production and secretion into culture media of IL-1B
(F),IL-6 (G), and CXCL8 (H) and co-treatment with nifedipine does not reduce this increased secretion.
Concurrent treatment with TNF and LPS does not increase secretion of MCP-1 (I) but does increase
secretionof IL-1B (J), IL-6 (K), and CXCL8 (L). Data is expressed as the median uorescence intensity (MFI)
of technical replicates as measured by Luminex assay normalised to the mass of tissue (mg). Treatments were
performed in duplicate and individual data points represent the mean of those technical replicates (n = 5
experiments). Statistical analysis was performed on the log2 transformation of the MFI/mg using an ANOVA
followed by Tukey’s multiple comparisons. e error bars represent SEM, * indicates P < 0.05, ** indicates
P < 0.01,*** indicates P < 0.001, and ns indicates no statistical dierence.
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environment with TNF and LPS induced myometrial cell contraction, which has been shown once previously15.
However, while these results strengthen the idea that targeting inammation may be therapeutically ecacious
in preventing preterm labour, further work is required.
Myometrial inammation can be either sterile (in the absence of pathogens) or microbial-associated and tends
to vary across gestation16. TNF and LPS have been used here to examine the activation of pathways involved in
both sterile and microbial inammation, respectively. LPS consistently and robustly increased myometrial cel-
lular and tissue inammation, signicantly upregulating pro-inammatory cytokines (mRNA and protein). In
response to pathogenic molecular components like LPS, myometrial cells produce cytokines such as IL-1B, IL-6,
CXCL8, and TNF which is enhanced by inltrating leukocytes such as macrophages, thus promoting a regulatory
positive feedback loop to maintain myometrial contractility9. Nifedipine did not reduce any marker of inamma-
tion we investigated, suggesting that its inhibitory action on smooth muscle contractions is not via regulation of
pro-inammatory cytokines. Conversely, TNF exposure for six hours did not induce expression or production of
key pro-inammatory cytokines. is may be a timing eect, but of note, LPS induced acute pro-inammatory
upregulation in the same time-frame, and as such may be due to inherent dierences in mechanisms of action.
ere is evidence in other tissues that nifedipine may have anti-inammatory properties beyond calcium
channel inhibition. For example, in human osteoarthritic chondrocytes, nifedipine at a concentration similar to
the one used here inhibited expression of IL-1B, IL-6, TNF, and cyclooxygenase-2, as well as inhibiting oxida-
tive stress13. Additionally, nifedipine is believed to have anti-inammatory and anti-oxidative stress eects on
endothelial cells14. erefore, while it was possible that nifedipine could have an anti-inammatory eect on
myometrial smooth muscle cells and tissue, results from this study do not support this hypothesis.
To recapitulate inammation associated with systemic infection, a known trigger of premature activation of
the myometrium, preterm labour was induced in an invivo mouse model using LPS. In our model, all mice gave
birth preterm within 24h of receiving LPS. is consistent timing of preterm birth ensured that any observed
delay in pregnancy was due to nifedipine and not to a variable eect of LPS. Nifedipine was unable to consistently
prolong gestation in this model, which contrasts to what is observed clinically, where a recent randomised control
trial showed that nifedipine treatment delayed preterm birth by 48h in almost 80% of cases17. However, while it
Figure7. Mice treated with nifedipine (NIF) delayed LPS-induced preterm birth in some mice but was not
consistent. (A) Timeline indicating the treatment time-points. Pregnant mice were injected intraperitoneal with
lipopolysaccharide (LPS; n = 24) or vehicle (phosphate-buered saline; PBS; n = 2) on gestational day (D)16.5.
Mice were then immediately administered either vehicle (ethanol; n = 5), 1mg/kg nifedipine (n = 9) or 10mg/
kg nifedipine (n = 10). Treatments were repeated every 24-h until birth. (B) Gestational length was calculated as
days post coitum. Each data point represents one dam and error bars represent SEM, ns denotes no signicant
statistical dierence between groups, and ** denotes P < 0.01.
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is dicult to determine the exact equivalent timeframe, the 12 to 24h delay in the mouse gestation observed in
this study can be approximated to be a few days to a week in human pregnancy. erefore, this mouse model can
recapitulate preterm birth, but does not completely mirror the eects of nifedipine seen in humans in the clinic.
is may be due to the dierences between human and mouse parturition which is a limitation of this study. is
highlights the benets of developing and employing a multi-faceted experimental approach as used in our study.
e mRNA expression of pro-inammatory or contraction-associated genes in the mouse uteri post-birth did
not dier between control mice that delivered preterm or mice that received nifedipine. However, Ccl2 expression
was downregulated in uteri from the dams in which nifedipine delayed the deliveries. Strong conclusions cannot
be drawn, however it is known in humans that CCL2 (also known as MCP-1) is upregulated by the myometrium
during labour18 so this raises interest in whether CCL2 could stand as an attractive candidate to target for a
therapy. To note, a limitation of the current study is that as the time between delivery of the pups and collection
of the uteri was not controlled for within the mouse cohort. To gain a better understanding of how these gene
proles may dier with nifedipine treatment, the precise time of birth would be informative to ensure similar
time between mouse uteri directly following labour.
Our study provides evidence that nifedipine does not reduce inammation that likely drives uterine con-
tractions via upstream pathways. is is the rst study to investigate the potential eect of nifedipine on pro-
inammatory pathways in human myometrium, an important consideration for any potential tocolytic. Without
anti-inammatory eects, nifedipine may not provide protection to the fetus against the detrimental eects
of preterm labour-associated inammation, but could be combined with an anti-inammatory agent for dual
therapy. erefore, in cases where nifedipine is used to delay delivery, this must be considered and highlights the
urgent need for eective therapeutics that can safely prolong gestation and quench inammation.
In our preterm birth mouse model, whereby preterm mouse fetuses are not viable and dicult to collect,
we were unable to assess if nifedipine had any eects in improving inammation in utero. Future studies could
investigate earlier timepoints of gestation post-nifedipine treatment to determine if there were still detrimental
inammatory eects on the fetuses, particularly on the fetal brain. A dual therapy of nifedipine and a known
anti-inammatory agent could also be investigated to determine if this counters the inammation associated with
preterm labour. To complement our work in the human myometrium, investigation of the eect of nifedipine
on human fetal membranes, the decidua and the placenta should be investigated to gain a more holistic under-
standing of nifedipine’s action on all the gestational tissues that work in unison to maintain uterine quiescence
and maternal immune tolerance during pregnancy.
e major strength of this study is the inclusion of multiple invitro, exvivo, and invivo models, which in
unison form a robust, multi-faceted research approach. e studies used in here build upon and enhance already
established experimental protocols giving the methodology of this study credibility. However, our adaptation of
the Danish Myo Technology (DMT) myograph to measure human myometrial tissue in this study is innovative,
highly sensitive and a superior method to traditional tissue bath set-ups. Previous studies in the preterm birth
research space oen rely on a single model to assess the eects of innovative therapies. Our pipeline allowed the
study of the eects of nifedipine at the cellular, whole tissue, and whole systems levels.
One consideration to note is the use of myometrial samples obtained from term pre-labour pregnancies and
not from preterm labouring pregnancies. erefore, the ndings from this study may not translate directly to
clinical settings and may not reect the same eect in labouring myometrium. However, by limiting our sample
collection to term pregnancies we were able to study the invitro eects of nifedipine in a homogenous and physi-
ologically normal study population. Additionally, in using oxytocin within the tissue baths, we could simulate
some conditions of labour.
When utilising whole myometrial tissue collected from patients, we cannot conclude that the eects are
caused directly by the main functional cells of the myometrium—the smooth muscle cells—but could be due
to multiple cell types working in concert. Additionally, the myometrial samples collected from the lower seg-
ment of the uterus during caesarean section does not uniformly represent all the cellular and tissue structures
that the entire uterus is composed of19. Further work could elucidate the contributions of the other cell types in
regulating the inammatory response.
We have demonstrated nifedipine blocks both spontaneous and, for the rst time, inammation-induced
uterine contraction invitro and exvivo. However, as preterm labour is a complex process, inhibiting muscular
contraction of the uterine myometrium may not be enough to prevent preterm birth. We have shown that nifedi-
pine does not reduce markers of inammation and may explain why nifedipine is unable to consistently perform
as a tocolytic clinically. erefore, nifedipine appears to treat the symptoms of preterm labour rather than the
cause, thus is limited as a preterm birth therapy. Our study highlights the crucial need for new therapeutics for
preterm birth that target the inammatory pathways upstream of myometrial contractions.
Methods
Drugs and chemicals. Nifedipine (N7634, Sigma-Aldrich, Missouri, USA) was reconstituted in sterile
ethanol with vortexing at 50mM and was stored in a paralm-sealed tube (to prevent evaporation) in the dark
at 4°C. E. Coli lipopolysaccharide (LPS; L2630, Sigma) was reconstituted in Dulbecco’s phosphate-buered
saline (dPBS; Gibco, ermoFisher Scientic, Victoria, Australia), aliquoted and stored at −20°C. Recombinant
human tumour necrosis factor alpha (TNF; Gibco; ermoFisher Scientic) was reconstituted in sterile water,
aliquoted and stored at −80°C. L-glutamine was prepared by dissolving 73mg L-glutamine (Sigma-Aldrich) in
sterile water then lter sterilising to make a 100 × stock solution for 1:100 dilution into media. TSL was prepared
by dissolving 10ug/ml transferrin, 25ng/ml sodium selenite, 10nmol/l linoleic acid in sterile water.
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Human myometrium tissue collection. Ethical approval for this study was obtained from the Mercy
Health Human Research Ethics Committee (2020-051). All methods were performed in accordance with the
National Health and Medical Research Council ethical guidelines. Pregnant individuals presenting to the Mercy
Hospital for Women, Heidelberg, Australia, gave informed written consent for myometrial tissue collection. A
two-to-three-centimetre diameter myometrial sample was excised from the lower portion of the non-labouring
(and non-induced) uterus at term caesarean sections, from n = 11 singleton pregnancies with no known compli-
cations, no prior history of preterm birth, and no medication use during pregnancy. Patient characteristics and
demographic data are shown in Table1. Samples were collected into cold phosphate buer saline (PBS; 137mM
NaCl, 10mM Na2HPO4, 1.8mM KH2PO4, 2.7mM KCl, pH 7.4) and processed within 30min of collection.
Human myometrial strip contractility myography. Myometrial tissue samples were transferred to
cold Krebs buer (120mM NaCl, 5mM KCl, 1.2mM MgSO4, 1mM KH2PO4, 25 mM NaHCO3, 11.1mM
D-Glucose, 2.5mM CaCl2) and dissected with a scalpel into strips 8mm long x 2mm wide x 1mm thick along
the longitudinal axis aligned with the direction of the muscle bres. Strips were mounted to individual organ
baths (820MS system, Danish Myo Technology, Hinnerup, Denmark) lled with 7mL Krebs buer. Within
each organ bath, one end of the muscle strip was clamped to a calibrated force transducer and the other end to a
micromanipulator, so that the tissue between the clamps was ~ 5mm long. Once myometrial strips were clamped
into the bath, a passive tension of 2mN was applied20. Each organ bath was continuously aerated with carbogen
(95% O2, 5% CO2) and maintained at a temperature of 37°C. Data were collected and analysed using LabChart
Pro Version v8 1.21.
Spontaneous rhythmic contractions typically initiated within two hours of mounting the tissue strips. Tissue
strips that failed to develop spontaneous contractions within two hours were challenged with a high potassium
salt solution (40mM KPSS; 85mM NaCl, 40mM KCl, 1.2mM MgSO4, 1mM KH2PO4, 25mM NaHCO3,
11.1mM D-Glucose, 2.5mM CaCl2) for two minutes. If the tissue was non-responsive, it was removed from the
bath and replaced with a new strip in fresh Krebs buer and then le to equilibrate and develop spontaneous
contractions.
In an additional set of experiments, spontaneously contracting myometrium was further stimulated with
1nM oxytocin in the bath to amplify contractility, based on the method of Arrowsmith and colleagues (2018)
and optimised for this assay20.
For each myometrial strip, a pre-treatment contraction baseline (one hour of stable basal contractions) was
established to serve as reference. Nifedipine stock solution (50mM in ethanol) or vehicle (ethanol) was diluted
1:500 in Krebs buer to an intermediate concentration of 100µM. en, 7 µL of this intermediate solution was
added to 7mL buer in the baths, to perform a 1:1000 dilution producing a nal concentration of nifedipine
at 0.1µM and a nal concentration of 0.0002% v/v ethanol. Dose response experiments were performed to
Table 1. Demographic characteristics of the pregnant patients. Continuous variables are shown as the
mean ± standard deviation; categorical variables are shown as the number of cases (%).
Characteristics Val u e
Maternal age (years) 32.8 ± 2.9
Maternal body mass index pre-pregnancy (kg/m2)21.7 ± 7.8
Maternal body mass index at delivery (kg/m2)28.5 ± 4.2
Gestational age (weeks) 38.6 ± 0.4
Fetal body weight at birth (g) 3565 ± 378
Ethnicity
Caucasian (Europe, Middle East, North Africa, Americas, Australia) 8 (72.7)
East Asian (China, Korea, Japan, South East Asia) 1 (9.1)
Central Asian (India, Pakistan, Nepal, Sri Lanka) 2 (18.2)
Gravidity n
1 0 (0)
2 3 (27.3)
3 3 (27.3)
4 2 (18.2)
5 2 (18.2)
6 1 (9.1)
Parityn
1 2 (18.2)
2 3 (27.3)
3 3 (27.3)
4 2 (18.2)
5 1 (9.1)
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determine the optimal concentration that elicits an inhibitory eect and prevents any eect of the ethanol as
vehicle. Myometrial strips were incubated with nifedipine or vehicle for 1h. Myometrial strips were then washed
thrice with fresh 37°C Krebs buer to remove all treatment (washout) and then allowed to re-equilibrate for one
hour before being challenged with 40mM KPSS to check responsiveness at completion of experiment.
To determine changes in myometrial contractility, the frequency (contractions per hour), amplitude (height of
peak), time to peak (time from initiation of contraction to max amplitude), and duration (width of single contrac-
tions) of contractions were measured using LabChart (v8, ADInstruments, Bella Vista, NSW, 2153, Australia).
Changes in these outputs were normalised to the basal (pre-treatment) spontaneous contractions of each strip:
the frequency, amplitude, time to peak and duration in the pre-treatment period were assigned 100% and these
outputs in the post-treatment period were calculated as a percentage of these baseline contractions. is relative
dierence was then calculated relative to the baseline contractions of the pre-treatment vehicle-treated strips.
Human myometrial smooth muscle cell line culture and expansion. An immortalised myome-
trial smooth muscle cell line, Pregnant Human Myometrial 1–41 (PHM1-41, ATCC, Virginia, USA), was cul-
tured in T75 asks containing Dulbecco’s modied eagle medium (DMEM; High glucose + Glutamax, Gibco;
ermoFisher Scientic) supplemented with 10% fetal calf serum (FCS; Gibco; ermoFisher Scientic), 2mM
L-glutamine, and 0.1mg/ml Geneticin (G-418; Roche Diagnostics, Victoria, Australia). Cells were expanded and
used no higher than passage 30 for invitro experiments.
Human myometrial smooth muscle cell contraction assay. Collagen gel contraction assays were
performed to assess the eect of nifedipine on the contractility of myometrial smooth muscle cells. Conuent
PHM1-41 cells were harvested with TryplE Express Enzyme (Gibco; ermoFisher Scientic), centrifuged at
150xg for 5–10min and resuspended in serum-free DMEM containing 1% TSL and 2mM L-glutamine.
Cultrex rat collagen I (R&D Systems, Minnesota, USA) was combined with sterile 10X dPBS, water, and 1N
sodium hydroxide on ice as described in the manufacturer’s manual to make a 3mg/ml collagen solution. Stock
TNF, LPS and nifedipine were diluted in their vehicles (water, PBS, and ethanol, respectively). Vehicles of each
agonist were accounted for in each treatment.
Cells at a density of 150,000 cells/ml were added to the collagen solution in a 2:1 ratio. To stimulate contrac-
tions, 1ng/ml TNF was added into this cell/collagen solution. Nifedipine (10µM) or vehicle control (ethanol)
was also added into the solution. Dose response experiments were used to determine the optimal concentration
of nifedipine to elicit an inhibitory response but was not cytotoxic. 500 μL of this cell/collagen mixture was
pipetted into wells of 24-well plates. e cells were embedded in the collagen gel by incubating the plate at 37°C
for one hour until the gels had solidied into discs. en, 500 μL of serum-free media containing 100ng/ml
LPS and 10µM nifedipine (or vehicle control) treatments were added to the wells. e gel discs were detached
from the well walls by gently running a pipette tip along the gel edges. e plates were incubated for 48h. A
cell-free collagen-gel only experiment was also performed to determine if there was any contraction inherent
to the collagen gel itself.
Images of the oating gel discs were taken at 0 h and 48h using a ChemiDoc Imaging System (BioRad, Cali-
fornia, USA). e size of the gels was determined by the area as measured using ImageJ soware (v1.53a, National
Institute of Health, Maryland USA). e gel area measurements at 48h were calculated as a percentage original
area of the gels at the initial timepoint (0h) for each independent experiment. Treatments were performed in
quadruplicate and the experiment was repeated thrice.
Myometrial cell inammatory response studies. PHM1-41 cells were seeded at a density of 40,000
cells/well into 24-well plates in DMEM supplemented with 10% FCS and 2mM L-glutamine (G-418 omitted as
per ATCC recommendation). Cells were incubated overnight at 37°C (5% CO2, 20% O2) to adhere to the plate.
Cells were then serum-starved by replacement of media with DMEM supplemented with 1% TSL and 2mM
L-glutamine and incubated for a further 16h. Following serum-starving, cells were pre-treated with either TNF
(0.1ng/ml), LPS (100ng/ml) or vehicle control in a low-serum media (DMEM supplemented with 2% FCS and
2mM L-glutamine) for two hours. Subsequently, this pre-treatment was removed and replaced with treatments
of TNF or LPS, with or without nifedipine (10µM) and incubated for another 24h. At completion, cells were
washed with PBS and frozen at −80°C with lysis solution (Sigma-Aldrich) in preparation for RNA extraction.
Cell viability assay. Cell viability was assessed for each dose of TNF, LPS, and nifedipine used. e MTS
assay (Promega, Madison WI, USA) was carried out in a 96-well plate as per manufacturer’s instructions. Optical
density at 490nm absorbance was measured using a BioRad X-Mark Microplate Spectrophotometer and Bio-
Rad Microplate Manager 6 soware.
Ex vivo myometrial tissue inammatory response. Myometrial tissue samples were dissected into
fragments of approximately 1–2mm size with any vasculature, decidua or scar tissue excised and excluded.
ree pieces of dissected tissue (combined weight 20–30mg) were placed into each well of a 24-well plate con-
taining treatments of combinations of TNF (1ng/ml), LPS (5 ng/ml), and nifedipine (10µM) or vehicle in
DMEM (supplemented with 1% antibiotic–antimycotic (Gibco; ermoFisher Scientic), 10% FCS, and 2mM
L-glutamine). Myometrial tissue was incubated at 37°C under 8% O2 and 5% CO2 for 6h. e tissue pieces were
collected, blotted, weighed, and stored in RNAlater at 4°C for 48h before snap-freezing in liquid nitrogen and
storage at −80°C.
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Cytokine secretion assay. A panel of human pro-inammatory cytokines and chemokines (IL-1β, IL-6,
IL-8 (CXCL8), monocyte chemoattractant protein (MCP)-1 and MCP-3) were simultaneously measured (multi-
plex) in the collected cell and tissue media supernatant using Milliplex MAP Luminex microbead assays (Merck
Millipore, Massachusetts, USA) as per manufacturer’s instructions. Technical duplicates from each cell culture
experiment were pooled prior to multiplexing. Samples were multiplexed in technical duplicate without dilu-
tion and data analysed on a Luminex (Bioplex-200, BioRad) instrument. Minimum detectable thresholds were
0.8pg/ml (IL-1β), 0.9pg/ml (IL-6), 0.4pg/ml (IL-8), 1.9pg/ml (MCP-1), and 3.8pg/ml (MCP-3). For evaluation
of protein expression by myometrial smooth muscle cells invitro results are expressed as mean uorescent inten-
sity. For protein expression of myometrial tissue cultured and treated exvivo, the mean uorescence intensity
was calculated per milligram of tissue cultured to control for dierences in tissue weight.
Mouse PTB model. Animal experiments were approved by the Austin Health Animal Ethics Committee
(A2020/05672) and followed the National Health and Medical Research Council ethical guidelines for the care
and use of animals for scientic purposes. All methods have been reported in accordance with the ARRIVE
guidelines (https:// arriv eguid elines. org). Five-week-old CBA x C57BL/6 (F1) female mice (n = 26) were sourced
from Animal Resources Centre (Western Australia, Australia). Mice were group-housed in conventional open-
top cages (18–22°C; 50% relative humidity), with a 12-h light/dark cycle, and food and water available adlibi-
tum. Female mice were acclimated to the new facility for 1 week before being mated overnight with stud F1
male mice. Pregnancy was conrmed by the presence of a copulatory plug the following morning, designated as
gestational day (D)0.5. On the morning of D16.5, pregnant mice (n = 24) received a 100µl intraperitoneal injec-
tion of 0.7µg/g LPS in PBS to induce preterm delivery. Mice were randomised and immediately following LPS
injection, mice received either a 20µl intraperitoneal injection of 1mg/kg (n = 9)or 10mg/kg(n = 10) nifedipine
in ethanol or neat ethanol as vehicle control (n = 5) (See Fig.7A). e gestational length (from mating to birth)
was recorded for each dam. Mice that had littered by the morning of D17.5, within 24h of LPS administration,
were considered to have delivered preterm. A subset of mice (n = 2) received only a 100µl intraperitoneal injec-
tion of PBS on D16.5 as control for term gestation length in these mice.
Dams were humanely killed via cervical dislocation the morning aer they littered. Both uterine horns were
dissected and a portion of each horn was collected into RNAlater. Uterine horns were stored at 4°C in RNAlater
for a minimum of 48h before snap freezing in liquid nitrogen and storage at −20°C. Investigators were not
blinded when administering I.P. injections, but were blinded when analysing gestational length of each mouse.
Reverse transcription and qRT‑PCR. Total RNA was extracted from cells, tissue, and mouse uterine
horns using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) according to the manufac-
turer’s instructions. Myometrial tissue and mouse uterine horns (maximum 40mg) were homogenised using a
tissue homogeniser (Omni International, Georgia, USA) prior to RNA extraction and a proteinase K digestion
(P4850, Sigma-Aldrich) was included. RNA was quantied using the Nanodrop ND 1000 spectrophotometer
(Nanodrop Technologies Inc, Delaware, USA) and then converted to cDNA using the High Capacity cDNA
Reverse Transcription Kit (Applied Biosystems, Massachusetts, USA) as per manufacturer’s guidelines.
Quantitative real-time polymerase chain reaction (qPCR) was performed to evaluate the eect of TNF, LPS,
and nifedipine treatment on expression of pro-inammatory and myometrial contraction-associated genes.
Predesigned TaqMan gene expression assays were used to quantify mRNA expression of these genes of interest
(listed in Table2). Gene expression was quantied by real time qPCR on the CFX384 (BioRad) using FAM-
labelled Taqman universal PCR mastermix (Applied Biosystems) with the following thermocycling conditions:
50°C for 2min; 95°C for 10min, 95°C for 15s, 60°C for 1min (40 cycles).
All cDNA samples were run in technical duplicates in the PCRs. Data were normalised to house-keeping
genes (YHWAZ for myometrial cells, TOP1 for primary myometrial tissue, and Polr2a for mouse uteri) as internal
Table 2. TaqMan gene expression assay IDs.
Gene Full name Species TaqMan ID
YHWAZ Tyrosine 3-monooxygenase/Tryptophan 5-monooxygenase activation protein zeta Human Hs01122454_m1
TOP1 DNA topoisomerase I Human Hs00243257_m1
IL-1β Interleukin-1 beta Human Hs01555410_m1
IL-6 Interleukin-6 Human Hs00174131_m1
CXCL8 CXC motif chemokine ligand-8 Human Hs00174103_m1
Polr2a RNA polymerase II subunit A Mouse Mm00839502_m1
Il-1b Interleukin-1 beta Mouse Mm00434228_m1
Il-6 Interleukin-6 Mouse Mm00446190_m1
Tnf Tumor necrosis factor Mouse Mm00443258_m1
Nlrp3 NLR family pyrin domain containing 3 Mouse Mm00840904_m1
Gja1 Gap junction alpha-1 Mouse Mm00439105_m1
Ccl2 C–C Motif chemokine ligand 2 Mouse Mm00441242_m1
Oxtr Oxytocin receptor Mouse Mm01182684_m1
Ptgs2 Prostaglandin-endoperoxide synthase 2 Mouse Mm00478374_m1
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controls. e stability of these reference genes was conrmed for each dierent tissue type and has been previ-
ously examined21. Ct data were analysed using the ΔΔCt method of analysis. Statistical analysis was performed
on the ΔCt values and data were then calculated and graphed as fold-change relative to the agonist treatment
(i.e. TNF or LPS) using the 2−ΔΔCt method.
Data availability
e datasets generated and analysed in the current study are available from the corresponding author on rea-
sonable request.
Received: 25 November 2022; Accepted: 6 March 2023
References
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Acknowledgements
e authors acknowledge clinical research midwives Gabrielle Pell, Rachel Murdoch, Genevieve Christophers,
Alison Abboud, Elizabeth Ellis, the obstetric clinical and midwifery sta and patients at the Mercy Hospital for
Women (Heidelberg) for provision of myometrial tissue.
Author contributions
B.M.A.; study design, methodology development, performed laboratory studies, data analysis, wrote the rst
dra and edited dra. N.K.B.; study design, methodology development, performed laboratory studies, and edited
manuscript. N.D.A.; methodology development and edited manuscript. S.B.; methodology development and
helped with data analysis. D.A.D., A.H.; methodology development, data analysis and editing of manuscript. S.T.,
T.J.K.; editing of nal manuscript. N.J.H.; study design, methodology development, overall supervision of project,
funding acquisition, and editing of manuscript. All authors provided input into the nal dra of the manuscript.
Funding
is study was funded by a Ferring Pharmaceuticals Research Grant and a Mercy Perinatal Research Grant.
e University of Melbourne Felix Meyer Scholarship provided stipend to B.M.A. Salary support was received
from the National Health and Medical Research Council Fellowships to T.J.K. (#1159261), S.T. (#1136418) and
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Vol.:(0123456789)
Scientic Reports | (2023) 13:5646 | https://doi.org/10.1038/s41598-023-31077-x
www.nature.com/scientificreports/
N.J.H. (#1146128). e funders had no role in study design, data collection, analysis, decision to publish or the
preparation of the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 31077-x.
Correspondence and requests for materials should be addressed to N.J.H.
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